The regulation of expression of the human beta globin gene is still not well understood. Ongoing studies in our laboratory (Project #Z01 DK 25045-04 LCB) focused on the 643 base pairs (bp) upstream from the human beta globin gene. A series of deletion analyses, followed by transient expression assays and chloramphenical acetyl transferase (CAT) assays revealed 3 upstream DNA regions of regulatory importance. Two regions (between -643 and -490, and between -338 and -233) were found to have a negative effect on gene expression, since their deletion increased the CAT activity, and they were defined as Negative Control Region I (NCR1) and NCR2 respectively. Deletion of a third region (between -233 and -185) was followed by a dramatic drop in CAT activity, suggesting a positive role of that segment, thus defined as Positive Control Region (PCR). While the other control regions are currently being investigated, we are focusing on the PCR. The positive control effect was detected in a human erythroid cell line (K562) but not in a mouse erythroleukemia (MEL) or a Chinese Hamster (R1610) cell lines, suggesting a human erythroid tissue specific activity, in contrast with the NCRs. This specificity, as well as the proximity of the PCR to NCR2 raise the possibility that PCR may contain an enhancer sequence, namely a DNA sequence capable of increasing the promoter activity and hence the gene expression in a position and orientation independent manner. This speculation is based on reports describing enhancers as tissue specific and controlled by close negative control elements which repress the gene expression in other tissues, while the repression is removed in the specific tissue enabling the gene to be expressed (prealbumin liver cells, insulin in beta cells, etc.). In an attempt to understand the PCR role, we are cloning it (100 bp) into two BamH1 sites of the -185 deleted form of the fusion plasmid p-beta-GLCAT. Transfection of hemin induced K562 cells with the new DNA recombinants may answer the question of the PCR as an enhancer.